Human hemoglobinopathies, such as beta-thalassemia and sickle-cell disease, are among the likely diseases amenable to gene therapy. The adeno-associated virus 2 (AAV2), a non-pathogenic human parvovirus, has gained attention as a potentially useful vector. Although significant progress has been made in understanding the initial steps in the AAV2 infection pathway, the precise role of the cellular proteins involved in these processes has not been elucidated. For example, cell surface heparan sulfate proteoglycan (HSPG) has been shown to be the primary receptor for AAV2 binding, but the mechanism of viral entry into the cell is not completely understood. Also, other serotypes, such as AAV4 and AAV5, have not been evaluated for hematopoietic stem cell transduction. Similarly, although a second human parvovirus, designated parvovirus B19, known to possess a remarkable tropism for human hematopoietic cells in the erythroid lineage, has been developed as a vector, the precise steps in the virus host cell-interaction are not fully understood. We have documented that in addition to HSPG as a receptor, AAV2 also requires a co-receptor, fibroblast growth factor receptor 1 (FGFR1), for successful infection. We have also documented that cell surface expression of erythrocyte P antigen, reported to be a receptor for parvovirus B19, is necessary but not sufficient for a successful infection by parvovirus B19. Using recombinant AAV2-, AAV4-, AAV5-, and parvovirus B19-globin vectors, we will test the following hypotheses: 1. Efficient transduction of primary human hematopoietic stem/progenitor cells can be mediated by AAV2, AAV4 and/or AAV5 vectors, 2. Efficient entry of parvovirus B19 in primary human hematopoietic cells is mediated by a putative cellular co-receptor, and 3. Parvovirus vectors will prove to be safe and effective for therapeutic correction of hemoglobinopathies in animal models in vivo. The following four Specific Aims will be pursued: 1. Elucidation of underlying mechanisms of differential transduction of primary human hematopoietic stem cells from bone marrow and umbilical cord blood by AAV2-, AAV4-, and AAV5-globin vectors. 2. AAV-mediated erythroid lineage-restricted expression of human globin genes in human hematopoietic cells in vitro, and therapeutic correction in homozygous beta-thalassemic mice in vivo. 3. Identification and characterization of the putative cellular co-receptor for efficient transduction of primary human hematopoietic progenitor cells by parvovirus B19-globin vectors. 4. AAV- and parvovirus B19-mediated transduction and long-term, regulated expression of human globin genes in hematopoietic progenitor cells in non-human primates in vivo. The knowledge gained from these studies will be applicable in further development of AAV and parvovirus B19 vectors and their optimal use in gene therapy of beta-thalassemia and sickle-cell disease.